Journal of Vector Ecology
Vol. 38, no. 1
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Role of species composition in malaria transmission by the Anopheles funestus group (Diptera: Culicidae) in Ghana Samuel K. Dadzie*, Ruth Brenyah1, and Maxwell A. Appawu Noguchi Memorial Institute for Medical Research, Department of Parasitology, Legon, Accra, Ghana,
[email protected] Kwame Nkrumah University of Science and Technology, School of Medical Sciences, Department of Clinical Microbiology, Kumasi, Ghana
1
Received 25 October 2012; Accepted 24 December 2012 ABSTRACT: Malaria remains a public health problem in Ghana, with Anopheles gambiae and Anopheles funestus as the predominant vectors. While much information exists on the species composition of An. gambiae, very little exists for An. funestus. This study was carried out to determine the species composition of An. funestus Giles populations from three ecological areas in Ghana and investigate their role in malaria transmission. Mosquitoes were collected using human landing and pyrethrum spray methods. A total of 10,254 Anopheles individuals were collected, out of which An. funestus constituted 53.6% (5,496). An. funestus sensu stricto (s.s.) and Anopheles lessoni were identified as the only members of the An. funestus group in all three ecological areas. All 62 sporozoite positive specimens that were identified as An. funestus s.s. were highly anthropophilic with a human blood index in the range of 80-96%, whereas more than 83% of the An. leesoni had fed on either bovine, goat, or sheep. Malaria transmission was higher in the Sahel savannah area than the rest of the ecological zones, with An. funestus s.s. being implicated as a vector of malaria in all ecological zones. Anopheles leesoni occurred in all the ecological areas but played no role in malaria transmission. The study established the importance of An. funestus s.s. in malaria transmission in Ghana. Journal of Vector Ecology 38 (1): 105-110. 2013. Keyword Index: Mosquitoes, Anopheles funestus, ecology, malaria, transmission, Ghana.
INTRODUCTION Anopheles funestus Giles is one of the two most important and widely distributed vectors of malaria in Africa (Gillies and de Meillon 1968). In some cases, its potential to transmit Plasmodium falciparum far outstrips that of Anopheles gambiae (Fontenille et al. 1997). It is also responsible for malaria epidemics in some areas (Fontenille et al. 1997). The species belongs to a group of nine morphologically similar but genetically different species (Gillies and Coetzee 1987). Recent findings have discovered ‘An. rivulorum’ species as a potential tenth species in the An. funestus group (Cohuet et al. 2003). An. funestus sensu stricto (s.s) play a major role in malaria transmission in many areas (Gillies and Coetzee 1987, Fontenille et al. 1997, Costantini et al. 1999, Cohuet et al. 2004) and to a lesser extent An. rivulorum Leeson Evans (Gillies and Coetzee 1987, Wilkes et al. 1996, Kawada et al. 2012) The other members of the group are mainly zoophilic (Gillies and Coetzee 1987). An. funestus group may be genetically differentiated into locally adapted populations throughout its range in tropical Africa with different vectorial and behavioral capacities (Boccolini et al. 1994, Lochoarn et al. 1998, Costantini et al. 1999). It is therefore important that members of the group are accurately identified so as to be able to understand their role in malaria disease transmission patterns and hence formulate evidence-based control strategies to reduce malaria transmission. An. gambiae Giles and An. funestus Giles are widely distributed in Ghana and have been implicated as the major vectors of malaria in many areas of the country (Appawu 2001a, 2004). Much information is available on species composition and the vectorial role of the various species of An. gambiae complex (Appawu et al. 2001a, 2004, Yawson et al. 2004) in the country. Anopheles funestus
has also been implicated as a vector of lymphatic filariasis in the coastal (Dunyo 1996) irrigated community (Dzodzomenyo et al. 1999) and in northern parts of Ghana (Appawu et al. 2001b). However, there is very little information on the species composition of An. funestus group across the different ecological areas in Ghana. Malaria vector control in the country will require more detailed knowledge of the genetic heterogeneities within populations of An. funestus and their effects on malaria transmission. The study determined the An. funestus group species composition from three ecological areas in Ghana and their biting and feeding behavior as well as the infection rates and entomological inoculation rates in order to better understand their epidemiologic role in malaria transmission. MATERIALS AND METHODS Mosquito collection sites and sampling methods Mosquitoes were collected in three ecological areas: Navrongo, in the Sahel savannah belt (10º 30’ N, 1º 30’ W), Ejura, in a forest (06º 40’ N, 09º 8.72’ W), and Dodowa, in the coastal forest belt (05º 52.673’ N, 000º 06.365’ W). The average annual rainfall in the Sahel savannah area is 850 mm, most of which occurs in the wet months of May-September with the rest of the year being relatively dry. A large reservoir in the middle of the district provides water for irrigation. The vegetation of the forest site is mainly deciduous and the inhabitants are mostly crop farmers and small scale traders. The area is characterized by a dense network of rivers and streams and receives an annual rainfall of about 2,000 mm. The coastal forest area is characterized by a secondary forest with grassland and small shrub trees. There are few streams and ponds, which usually dry out during the dry season. Tree cover in
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the area is, however, rapidly decreasing as a result of farming and economic activities and has led to deforestation. Average rainfall ranges between 350 and 1,200 mm annually. Biting and indoor resting adult female An. funestus mosquitoes were collected from the different ecological areas in Ghana using human landing catches and pyrethrum spray collections, respectively (WHO 1975). A total of 32 human-nights of human landing catches (LC) were carried out indoors and outdoors of rooms every month by two teams of four collectors. The wet season and dry season collections were done twice weekly in the month of June, 2001 and in January, 2002, respectively. Mosquitoes attempting to bite the collectors were collected and kept in paper cups covered by mesh screen. Collections were made from 18:00 to 06:00 and the collectors were rotated between indoors and outdoors after each hour of collection to compensate for differences in the individual mosquito attraction or repulsion. The study received ethical approval from the Noguchi Institutional Review Board. The mosquito collectors were offered treatment in case of any infection. The pyrethrum spray collection (PSC) was carried out between 06:00–09:00. This involved spreading white sheets on the floor of rooms and after closing all the windows, spraying with pyrethrum insecticide. After about 10 min, the white sheets were inspected and all mosquitoes knocked down or dead were collected. Mosquito processing Mosquitoes captured by landing catches were anaesthetized with ether, sorted, and morphologically identified into species (Gilles and de Meillon 1968) and counted. All the anophelines caught were stored in 1.5 ml Eppendorf snap vials with desiccants for further laboratory analysis. Heads and thoraces of a sample of An. funestus were removed and tested for the presence of circumsporozoite antigens (CS) of P. falciparum using the enzymelinked immunosorbent assay (ELISA) described by Wirtz et al. 1987. The abdomen of blood-fed An. funestus individuals were analyzed to identify the source of blood meal using the ELISA method (Beier et al. 1988). Genomic DNA was extracted from the legs of individual An. funestus using the method described by Scott et al. 1993. The extracted DNA was used as a template for PCR. The DNA was analyzed to identify the species of An. funestus using the cocktail PCR protocol and primers described by Koekemoer et al. (2002). PCR products were electrophoresed in 2.5% agarose gel, stained with ethidium bromide, and visualized on a UV-light.
June 2013
RESULTS Species composition of An. funestus group A total of 10,254 Anopheles mosquitoes was collected from the three ecological areas. Of the total, An. funestus constituted 53.6% (5,496), followed by An. gambiae s.l. at 46.1% (4,725), and An. rufipes, 0.3% (33). The Sahel savannah area yielded 80% (4,397) of the total number of An. funestus captured by both landing and pyrethrum spray methods, followed by 10.8% (594) from the coastal forest and 9.2% (505) from the forest area. Indoor biting collections from all the ecological zones made up 61.1% (767) of the collections while outdoor collections constituted 38.9% (490). Out of 812 (comprising 281 from Sahel savannah area, 263 from forest, and 268 from coastal forest) An. funestus mosquitoes analyzed, PCR amplification of the genomic DNA confirmed 98.3% (798) of the morphologically identified An. funestus as belonging to the An. funestus group. Further amplification and analysis of the ribosomal DNA showed that An. funestus s.s. constituted more than 90.6% (711) of all the biting and indoor resting catches in all the study areas while An. leesoni made up 9.4% (74). An. funestus s.s. constituted more than 95% and 81% of the LC and PSC, respectively, in all the three ecological areas (Table 1). An. leesoni also formed less than 5% and 8% of the LC and PSC, respectively (Table 1). Feeding behavior Biting rates of 13.0, 3.3, and 5.5 bites/human/night were estimated for An. funestus s.s in the Sahel savannah, forest, and coastal forest zones, respectively. The pattern of indoor and outdoor night-biting in the Sahel savannah area showed that biting rose progressively through the night and peaked during 05:00-06:00 GMT (Figure 1A). Two major peaks were observed in the forest area at 22:00-23:00 GMT and 24:00-01:00 GMT (Figure 1B) and a similar trend was observed in the coastal forest area (Figure 1C). Sporozoite infectivity and blood meal source A total of 812 An. funestus specimen from the three ecological areas were assayed using ELISA for the presence of P. falciparum circumsporozoite proteins. A total of 62 (7.6%) specimens of the An. funestus group tested positive for circumsporozoite proteins. Further analysis of the 62 positive samples by PCRs showed that all were An. funestus s.s. None of the An. leesoni tested positive for circumsporozoite proteins. A P. falciparum sporozoite rate of 5.0% (31/623) was estimated for An. funestus s.s. in the Sahel
Table 1. Percentage of An. funestus individuals collected from Sahel savannah, forest, and coastal forest areas by different collection methods. Ecological site/Community
Night Biting Catches No. examined 140
% An. funestus s.s. 97.5
Forest/Ejura
130
Coastal forest/ Dodowa
134
Total
404
Sahel savannah/ Navrongo
Pyrethrum Spray Collection Catches
2.5
No. examined 141
% An. funestus s.s. 91.6
95.4
4.6
133
95.7
4.3
98.5
1.5
134
97.3
2.7
% An. leesoni
408
% An. leesoni 8.4
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A
B
Figure 2. The entomological inoculation rates (EIR, infective bites per human-night) of An. funestus s.s. captured from the Sahel savannah, forest, and coastal forest areas in the wet and dry seasons. DISCUSSION
C
Figure 1. Human night biting pattern of An. funestus s.s. captured from A) Sahel savannah, B) forest, and C) coastal forest areas.
savannah area compared to 4.1% (13/318) and 4.7% (18/381) in the forest and coastal forest areas, respectively. A significantly higher (P
